• Journal of Animal Reproduction and Biotechnology
• /
• v.35 no.4
• /
• pp.289-296
• /
• 2020

Spermatogonial stem cells are self-renewal and differentiate into sperm in post-pubertal mammals. There exists a balance between the self-renewal and differentiation in the testes. Spermatogonial stem cells make up only 0.03% of testicular cells in adult mice. These cells maintain sperm production by differentiating after puberty. Therefore, analyzing the expression of genes associated with spermatogenesis is critical for understanding differentiation. The present study aimed to establish the postnatal period of cells in relation to spermatogenesis. To study the expression of differentiated and undifferentiated marker genes in enriched spermatogonial stem cells, in vitro culture was performed and cells from pup (6-8-day-old) and adult (4-months-old) testicular tissues were isolated. As a result, undifferentiated genes, Pax7, Plzf, GFRa1, Etv5 and Bcl6b, were highly increased in cultured spermaotogonial stem cells compared with pup and adult testicular cells. On the other hands, differentiated gene, c-kit was highly increased in adult testicular cells, Also Stra8 gene was highly increased in pup and adult testicular cells. This study provides a better understanding of spermatogenesis-associated gene expression during postnatal periods.

• Journal of Animal Science and Technology
• /
• v.65 no.1
• /
• pp.160-174
• /
• 2023

The purpose of this study was to compare marbling score, meat quality, juiciness, sarcomere length, and skeletal muscle satellite cell (SMSC) growth and related gene expression between Woori black pig (WB) and the Landrace, Yorkshire, and Duroc (LYD) crossbreed at different body weights (b.w.). WB was developed to improve meat quality and growth efficiency by crossbreeding Duroc with Korean native black pig. A total of 24 pigs were sacrificed when their b.w. reached about 50, 75, 100, and 120 kg. SMSC were isolated from the femoris muscles, and muscle and adipose tissues were sampled from the middle and the subcutaneous part of the femoris of hind legs, respectively. Expression levels of genes including Myoblast determination protein 1 (MyoD), Paired box gene 3 (Pax3), Myosin heavy chain (MyHC), and Myogenin, which are responsible for the growth and development of SMSC, were higher in LYD than the WB. Muscle growth inhibitor myostatin (MSTN), however, was expressed more in WB compared to LYD (p < 0.01). Numbers of SMSC extracted from femoris muscle of LYD at 50, 75, 100, and 120 kg b.w. were 8.5 ± 0.223, 8.6 ± 0.245, 7.2 ± 0.249, and 10.9 ± 0.795, and those from WB were 6.2 ± 0.32, 6.2 ± 0.374, 5.3 ± 0.423, and 17.1 ± 0.315, respectively. Expression of adipogenic genes in adipose tissue including CCAAT/enhancer-binding protein (CEBP)-β, peroxisome proliferator activated receptor (PPAR)-γ, and fatty acid synthase (FASN), were greater in WB when compared with LYD (p < 0.01). Results from the current study suggest that different muscle cell numbers between 2 different breeds might be affected by related gene expression and this warrants further investigation on other growth factors regulating animal growth and development.

• Journal of Animal Science and Technology
• /
• v.65 no.3
• /
• pp.664-678
• /
• 2023

To improve culture efficiency of Hanwoo myosatellite cells, these cells were cultured at different temperatures. Hanwoo myosatellite cells were compared with C2C12 cells to observe proliferation and differentiation at culture temperatures of 37℃ and 39℃ and determine the possibility of using them as cultured meat. Immunofluorescence staining using Pax7 and Hoechst, both cells cultured at 37℃ proliferated better than cultured at 39℃ (p < 0.05). When differentiated cells were stained with myosin and Hoechst, there was no significant difference in myotube thickness and Fusion index (p > 0.05). In Western blotting analysis, Hanwoo myosatellite cells were no significant difference in the expression of myosin between cells differentiated at the two temperatures (p > 0.05). C2C12 cells were no significant difference in the expression of myosin between cells differentiated at the two temperatures (p > 0.05). In reverse transcription and quantitative polymerase chain reaction (RT-qPCR) analysis, Hanwoo myosatellite cells cultured at 39℃ had significantly (p < 0.05) higher expression levels of MyHC, MYF6, and MB than those cultured at 37℃. C2C12 cells cultured at 39℃ showed significantly (p < 0.05) higher expression levels of MYOG and MB than those cultured at 37℃. To increase culture efficiency of Hanwoo myosatellite cells, proliferating at 37℃ and differentiating at 39℃ are appropriate. Since results of temperature differences of Hanwoo myosatellite cells were similar to those of C2C12 cells, they could be used as a reference for producing cultured meat using Hanwoo satellite cells.

• Food Science of Animal Resources
• /
• v.43 no.6
• /
• pp.1017-1030
• /
• 2023

Fetal bovine serum (FBS), which contains various nutrients, comprises 20% of the growth medium for cell-cultivated meat. However, ethical, cost, and scientific issues, necesitates identification of alternatives. In this study, we investigated commercially manufactured serum-free media capable of culturing Hanwoo satellite cells (HWSCs) to identify constituent proliferation enhancing factors. Six different serum-free media were selected, and the HWSC proliferation rates in these serum-free media were compared with that of control medium supplemented with 20% FBS. Among the six media, cell proliferation rates were higher only in StemFlex^TM Medium (SF) and Mesenchymal Stem Cell Growth Medium DXF (MS) than in the control medium. SF and MS contain high fibroblast growth factor 2 (FGF2) concentrations, and we found upregulated FGF2 protein expression in cells cultured in SF or MS. Activation of the fibroblast growth factor receptor 1 (FGFR1)-mediated signaling pathway and stimulation of muscle satellite cell proliferation-related factors were confirmed by the presence of related biomarkers (FGFR1, FRS2, Raf1, ERK, p38, Pax7, and MyoD) as indicated by quantitative polymerase chain reaction, western blotting, and immunocytochemistry. Moreover, PD173074, an FGFR1 inhibitor suppressed cell proliferation in SF and MS and downregulated related biomarkers (FGFR1, FRS2, Raf1, and ERK). The promotion of cell proliferation in SF and MS was therefore attributed to FGF2, which indicates that FGFR1 activation in muscle satellite cells may be a target for improving the efficiency of cell-cultivated meat production.

• Food Science of Animal Resources
• /
• v.44 no.3
• /
• pp.710-722
• /
• 2024

Extracellular matrix (ECM) proteins play a crucial role in culturing muscle stem cells (MuSCs). However, there is a lack of extensive research on how each of these proteins influences proliferation and differentiation of MuSCs from livestock animals. Therefore, we investigated the effects of various ECM coatings-collagen, fibronectin, gelatin, and laminin-on the proliferation, differentiation, and maturation of porcine MuSCs. Porcine MuSCs, isolated from 14-day-old Berkshire piglets, were cultured on ECM-coated plates, undergoing three days of proliferation followed by three days of differentiation. MuSCs on laminin showed higher proliferation rate than others (p<0.05). There was no significant difference in the mRNA expression levels of PAX7, MYF5, and MYOD among MuSCs on laminin, collagen, and fibronectin (p>0.05). During the differentiation period, MuSCs cultured on laminin exhibited a significantly higher differentiation rate, resulting in thicker myotubes compared to those on other ECMs (p<0.05). Also, MuSCs on laminin showed higher expression of mRNA related with maturated muscle fiber such as MYH1 and MYH4 corresponding to muscle fiber type IIx and muscle fiber type IIb, respectively, compared with MuSCs on other ECM coatings (p<0.05). In summary, our comparison of ECMs revealed that laminin significantly enhances MuSC proliferation and differentiation, outperforming other ECMs. Specifically, muscle fibers cultured on laminin exhibited a more mature phenotype. These findings underscore laminin's potential to advance in vitro muscle research and cultured meat production, highlighting its role in supporting rapid cell proliferation, higher differentiation rates, and the development of mature muscle fibers.

• Asian Pacific Journal of Cancer Prevention
• /
• v.16 no.18
• /
• pp.8533-8539
• /
• 2016

Background: B-cell chronic lymphocytic leukemia B (B-CLL), the most common type of leukemia, may be caused by apoptosis deficiency in the body. Adipose tissue-derived mesenchymal stem cells (AD-MSCs) as providers of pro-apoptotic molecules such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), can be considered as an effective anti-cancer therapy candidate. Therefore, in this study we assessed the role of tumor necrosis factor-producing mesenchymal stem cells oin apoptosis of B-CLL cells resistant to fludarabine-based chemotherapy. Materials and Methods: In this study, after isolation and culture of AD-MSCs, a lentiviral LeGO-iG2-TRAIL-GFP vector containing a gene producing the ligand pro-apoptotic with plasmid PsPAX2 and PMDG2 virus were transfected into cell-lines to generate T293HEK. Then, T293HEK cell supernatant containing the virus produced after 48 and 72 hours was collected, and these viruses were transduced to reprogram AD-MSCs. Apoptosis rates were separately studied in four groups: group 1, AD-MSCs-TRAIL; group 2, AD-MSCs-GFP; group 3, AD-MSCs; and group 4, CLL. Results: Observed apoptosis rates were: group 1, $42{\pm}1.04%$; group 2, $21{\pm}0.57%$; group 3, $19{\pm}2.6%$; and group 4, % $0.01{\pm}0.01$. The highest rate of apoptosis thus occurred ingroup 1 (transduced TRAIL encoding vector). In this group, the average medium-soluble TRAIL was 72.7pg/m and flow cytometry analysis showed a pro-apoptosis rate of $63{\pm}1.6%$, which was again higher than in other groups. Conclusions: In this study we have shown that tumor necrosis factor (TNF) secreted by AD-MSCs may play an effective role in inducing B-CLL cell apoptosis.

• Clinical and Experimental Reproductive Medicine
• /
• v.36 no.1
• /
• pp.23-34
• /
• 2009

Objectives: Mesenchymal stem cells (MSC) comprise a promising tool for cellular therapy. It is known that long-term in vitro culture of human bone marrow and adipose tissue derived-MSCs lead to a reduction of life span and a change of stem-like characters. The aim of our study was to examine whether stem cell properties of human umbilical cord-derived stem cells (HUC) could be affected by in vitro expansion. Methods: HUC were isolated from human umbilical cord and cultured for 10 passages in vitro. Morphology and population doubling time (PDT) were investigated, and changes of stem cell properties were examined using RT-PCR and immunocytochemistry during serial subcultures. Results: Morphology and PDT of HUC began to change slightly from the 7th passage (p7). Expression level of nestin and vimentin mRNAs increased along with the culture period from p4 until p10. In contrast, expression level of SCF mRNA decreased during the same culture period. Expression level of Oct-4 and HNF-4${\alpha}$ mRNAs was not significantly changed throughout the culture period until p10. Expression level of BMP-4, FGF-5, NCAM and HLA-ABC mRNAs appeared to increase as the culture continued, however, the difference was not significant. Immunocytochemical studies showed that HUC at p3, p6 and p9 positively were stained with antibodies against SSEA-3 and SSEA-4 proteins. Interestingly, staining intensity of HUC for ICAM-1 and HLA-ABC gradually increased throughout the culture period. Intensity against thy-1 and fibronectin antibodies increased at p9 while that against TRA-1-60 and VCAM-1 antibodies began to decrease at p6 until p9. Conclusions: These results suggest that HUC change some of their stem cell characteristics during in vitro culture. Development of culture system might be needed for the maintenance of characteristics.

• Development and Reproduction
• /
• v.12 no.1
• /
• pp.87-95
• /
• 2008

Neural stem/precursor derived from pluripotent human embryonic stem cells (hESCs) has considerable therapeutic potential due to their ability to generate various neural cells which can be used in cell-replacement therapies for neurodegenerative diseases. However, production of neural cells from hESCs remains technically very difficult. Understanding neural-tube like rosette characteristic neural precursor cells from hESCs may provide useful information to increase the efficiency of hESC neural differentiation. Generally, neural rosettes were derived from differentiating hEBs in attached culture system, however this is time-consuming and complicated. Here, we examined if neural rosettes could be formed in suspension culture system by bypassing attachment requirement. First, we tested whether the size of hESC clumps affected the formation of human embryonic bodies (hEBs) and neural differentiation. We confirmed that hEBs derived from $500{\times}500\;{\mu}m$ square sized hESC clumps were effectively differentiated into neural lineage than those of the other sizes. To induce the rosette formation, regular size hEBs were derived by incubation of hESC clumps($500{\times}500\;{\mu}m$) in EB medium for 1 wk in a suspended condition on low attachment culture dish and further incubated for additional $1{\sim}2$ wks in neuroectodermal sphere(NES)-culture medium. We observed the neural tube-like rosette structure from hEBs after $7{\sim}10$ days of differentiation. Their identity as a neural precursor cells was assessed by measuring their expressions of neural precursor markers(Vimentin, Nestin, MSI1, MSI2, Prominin-1, Pax6, Sox1, N-cadherin, Otx2, and Tuj1) by RT-PCR and immunofluorescence staining. We also confirmed that neural rosettes could be terminally differentiated into mature neural cell types by additional incubation for $2{\sim}6$ wks with NES medium without growth factors. Neuronal(Tuj1, MAP2, GABA) and glial($S100{\beta}$ and GFAP) markers were highly expressed after $2{\sim}3$ and 4 wks of incubation, respectively. Expression of oligodendrocyte markers O1 and CNPase was significantly increased after $5{\sim}6$ wks of incubation. Our results demonstrate that rosette forming neural precursor cells could be successfully derived from suspension culture system and that will not only help us understand the neural differentiation process of hESCs but also simplify the derivation process of neural precursors from hESCs.

• Journal of Korean Society of Forest Science
• /
• v.62 no.1
• /
• pp.24-42
• /
• 1983

To develop Ilex cornuta which grow naturally in the southwest seaside district as new ornamental tree, the author chose I. cornuta growing in the four natural communities and those cultivated in Kwangju city as a sample, and investigated its ecology, morphology and characteristics. The results obtained was summarized as follows; 1) The natural distribution of I. cornuta marks $35^{\circ}$43'N and $126^{\circ}$44'E in the southwestern part of Korea and $33^{\circ}$20'N and $126^{\circ}$15'E in Jejoo island. This area has the following necessary conditions for Ilex cornuta: the annual average temperature is above $12^{\circ}C$, the coldness index below $-12.7^{\circ}C$, annual average relative humidity 75-80%, and the number of snow-covering days is 20-25 days, situated within 20km of from coastline and within, 100m above sea level and mainly at the foot of the mountain facing the southeast. 2) The vegetation in I. cornuta community can be divided that upper layer is composed of Pinus thunbergii and P. densiflora, middle layer of Eurya japonica var. montana, Ilex cornuta and Vaccinium bracteatum, and the ground vegetation is composed of Carex lanceolata and Arundinella hirta var. ciliare. The community has high species diversity which indicates it is at the stage of development. Although I. cornuta is a species of the southern type of temperate zone where coniferous tree or broad leaved, evergreen trees grow together, it occasionally grows in the subtropical zone. 3) Parent rock is gneiss or rhyolite etc., and soil is acidic (about pH 4.5-5.0) and the content of available phosphorus is low. 4) At maturity, the height growth averaged $10.48{\pm}0.23cm$ a year and the diameter growth 0.43 cm a year, and the annual ring was not clear. Mean leaf-number was 11.34. There are a significant positive correlation between twig-elongation and leaf-number. 5) One-year-old seedling grows up to 10.66 cm (max. 18.2 cm, min. 4.0 cm) in shoot-height, with its leaf number 12.1 (max. 18, min), its basal diameter 2.24 mm (max. 4.0 mm, min. 1.0 mm) and shows rhythmical growth in high temperature period. There were significant positive correlations between stalk-height and leaf-number, between stalk-height and basal-diameter, and between number and basal diameter. 6) The flowering time ranged from the end of April to the beginning of May, and the flower has tetra-merouscorella and corymb of yellowish green. It has a bisexual flower and dioecism with a sexual ratio 1:1. 7) The fruit, after fertilization, grows 0.87 cm long (0.61-1.31 cm) and 0.8 cm wide (0.62-1.05 cm) by the beginning of May. Fruits begin to turn red and continue to ripen until the end of October or the beginning of November and remain unfading until the end of following May. With the partial change in color of dark-brown at the beginning of the June fruits begin to fall, bur some remain even after three years. 8) The seed acquision ratio is 24.7% by weight, and the number of grains per fruit averages 3.9 and the seed weight per liter is 114.2 gram, while the average weight of 1,000 seeds is 24.56 grams. 9) Seeds after complete removal of sarcocarp, were buried under ground in a fixed temperature and humidity and they began to develop root in October, a year later and germinated in the next April. Under sunlight or drought, however, the dormant state may be continued.

• Journal of Korean Society of Forest Science
• /
• v.42 no.1
• /
• pp.1-38
• /
• 1979

The woody species of Genus Ilex which are endemic to Korea are distributed on limited area due to solely temperature factor. There is some differences according to species, however in general, the evergreen Ilex are found along southern coastal area of Korean Peninsula and near islands where the cold index does not exceed $-5^{\circ}C$. But Ilex macropoda and the variety, only deciduous ones, are grown in temperate zone of the peninsula and some islands. The list of Ilex species of Korea are as follows. Ilex cornuta Lindley et Pax., I. crenata Thunb. var. microphylla Max., I. crenata Thunb., I. rotunda Thunb., I. macropoda Miq., I. macropoda Miq. var. pseudo-macropoda Loensner, I. integra Thunb. The author surveyed the populations of Ilex species as many as possible and data of some characters such as leaf shape, spine, fruit shape, stomata density, sex ratio in natural communities, etc. are collected. Almost all the Ilex species in Korea show sporadic distribution. This means quite small sized populations isolate distantly each other eliminating the change of gene exchange in between. Particularly Ilex conuta and I. crenata show the morphological differentiation among populations as well as significant individual variation within a population. These were true with such characteristics, leaf shape, leaf dimension, leaf margin, fruit shape, spine, and stomata density. The founded are that the fruit length and the stomata density counted on the beneath surface of leaves of Ilex cornuta increased with the decrease of latitude. These are naturally closely related with the cold index values. The table shown below indicates the correlation between mean stomata density per $0.3642mm^2$ and cold index values. These relation however were not observed on Ilex crenata. The most dominated natured in relation to individual variation were outline of leaf, the number of marginal spine, the shape of leaf cross section and the degree of luster of the upper leaf surface. As shown in photos 5~7, these variations are agreed at a glance. There are reports that the development of marginal spines in some Ilex species is associated with the juvenility and topophysis. In present study, these two factors were neglected because of the intended sampling procedure. Of Ilex rotunda, population difference with the characteristics of leaf length is recognized but not for leaf width, petiole length, and fruit size. However, individual variations within a population were significantly large. In case of Ilex integra, only individual differences within population were calculated statistically for such characteristics as leaf length, leaf width, and petiole length. As to natural population, the sex ratio was 1:2 (female to male) for Ilex cornuta, and 1:1 for Ilex crenata. The tendency of more male than female in I. cornuta was agreed to other observations. Preparing the tip cutting of length 10cm, and treating with IBA, then attaching earth ball to the cut end, very successful rooting percentages were obtained. Asexual propagation has the advantages of maintaining the heterozygosity of existing varieties and overcoming the difficulties of delayed seed germination frequently encountered with Ilex species. Considering a great deal of variation in morphological traits, a good possibility of selection breeding for decorative and ornamental purposes exists. At present, these evergreen Ilex are ignored by local people as nuisance weedy shrubs. So the proper protection measures should promptly be taken.